Extended Data Fig. 3: Nascent and parental MCMs are equally proficient in pre-RC formation.
From: Equilibrium between nascent and parental MCM proteins protects replicating genomes
a, Top, dual-HaloTag labelling protocol in MCM4–Halo cells. Middle and bottom, QIBC of MCM4–Halo cells stained for parental and nascent MCM4 and DAPI after pre-extraction. The dashed box marks the cell doubling time; n ≈ 2,000 cells per condition. b, Top, MCM4–Halo dual labelling protocol. Middle and bottom, QIBC of cells transfected with control siRNA or siRNA against CDC6 and stained for chromatin-bound parental and nascent MCM4 and DAPI. n ≈ 9,500 cells per condition. c, Top, QIBC-based sub-stratification of G1 phase by CDT1 immunostaining in pre-extracted MCM4–Halo cells; DAPI indicates DNA content. Bottom, quantification of chromatin bound parental and nascent MCM4 at the indicated G1 stages defined at the top and according to the labelling protocol depicted in b. Each data point indicates mean ± s.d. of mean intensity of parental and nascent MCM pools normalized individually as 100% with respect to the G1-phase gate 7; n = 3 biological replicates with n ≈ 4,500 cells per replicate. d, Left, dual-HaloTag labelling protocol in MCM2–Halo cells. Middle and right, QIBC of MCM2–Halo cells stained for parental and nascent MCM2 without pre-extraction. The dashed box marks the cell doubling time. Lines denote medians; n ≈ 4,400 cells per condition. e, Top and bottom, QIBC of MCM2–Halo cells stained for parental and nascent MCM2 and DAPI after pre-extraction according to the labelling protocol depicted in d. The dashed box marks the cell doubling time; n ≈ 3,800 cells per condition.
